Department of Microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharashtra, India
Abstract
Background and objective: Urinary tract infection(UTI) is one of the most common bacterial infections encountered in clinical practice. UTIs caused by extended-spectrum beta-lactamase (ESBL) AmpC and metallo-beta-lactamase (MBL) producing Escherichia coli (E. coli) are difficult to treat. Fosfomycin is an old antibiotic that has excellent bactericidal activity against a wide range of bacteria. This study aimed to determine the fosfomycin susceptibility of E. coli causing UTI in a tertiary care hospital in Western Maharashtra, India.
Material and methods: The study was conducted at a tertiary care center in Pune, a city of Western Maharashtra, India. Urine samples from UTI cases yielding significant (> 1x 105 cfu/ml) growth of E. coli were included in study. E. coli isolates were tested for susceptibility to fosfomycin and a panel of antimicrobial agents by Kirby Bauer disc diffusion method. All the isolates were tested for production of ESBL, AmpC and MBL.
Result: A total of 88 E. coli were isolated of which, 47 (53.40%) and 41 (46.59%) were from male and female patients respectively. Of the total E. coli isolates, 58 (65.9%) were from in-patient cases. Multi-drug resistance was found in 69 (78.40%) isolates and remaining 19 (21.6%) were resistant to different antimicrobials tested. All (100%) the MDR and non-MDR isolates were sensitive to fosfomycin. Highest resistance was present against nalidixic acid (93.8%) while resistance was least against nitrofurantoin (15.91%), piperacillin/tazobactam (17.1%) and meropenem (18.18%). Of the total, 35 (50.72%) isolates were both AmpC and ESBL producers while 11 (15.94%) and 8 (11.59%) were only AmpC and ESBL producers respectively. MBL was positive in 15 (21.73%) of E. coli isolates. All those isolates tested sensitive to fosfomycin.
Conclusion: The study revealed that fosfomycin had excellent activity against MDR E. coli causing UTI in our area.
IMC J Med Sci. 2023; 17(1): 008. DOI: https://doi.org/10.55010/imcjms.17.008
*Correspondence: Dr. Sameena Khan, Department of Microbiology, Dr. D. Y. Patil Medical College, Hospital and Research Centre, Dr. D. Y. Patil Vidyapeeth, Pimpri, Pune, Maharashtra, India. E-mail: [email protected]
Introduction
Urinary tract infection(UTI) is a common bacterial infection of urinary system and requires antibiotics for treatment [1]. Beta-lactams, co-trimoxazole, fluoroquinolones and other antimicrobial agents have been used for many years in the treatment of UTI. But UTI caused by emerging multi-drug resistant and extended-spectrum beta-lactamases (ESBLs), AmpC and metallo-beta-lactamase (MBL) producing organisms has made treatment of UTI difficult and expensive. Fosfomycin is an old bactericidal agent which has a good in vivo and in vitro activity against a wide range of bacteria and thus making it a good option for the treatment of UTI [2-4]. Fosfomycin also shows very good activity in penetrating biofilms of Gram-negative bacteria in monotherapy as well as in combined therapy and has very good eradication activity [5]. The main mechanism by which fosfomycin acts is by irreversibly inhibiting the bacterial cell wall biosynthesis. After entering into cytoplasm of bacteria, fosfomycin binds with MurA enzyme and inhibits peptidoglycan biosynthesis [3,6]. Apart from being effective, fosfomycin formulations have less adverse effects than other antimicrobial agents. Mild gastro intestinal distress is the most commonly reported adverse effect [7]. Therefore, this study was undertaken to assess the fosfomycin susceptibility of Escherichia coli causing UTI in a tertiary care center in Western Maharashtra, India. Results of the study would help in guiding treatment of UTIs due to sensitive as well as multi-drug resistant (MDR) pathogens.
Material and Methods
The study was carried out at a tertiary care center based in Pune, a city of Western Maharashtra, India. It was approved by the Institutional Ethical Sub-committee (Letter number: IESC/30/2022 dated: 17 February 2022).
Urine samples from in and out patient departments having clinical features of UTI were collected and included in the study. Samples yielding significant (> 1x 105 cfu/ml) [8] growth of E. coli were included in study for further analysis. Any urine sample which yielded a non-significant count and organisms other than E. coli was excluded from the study. Relevant patient-related demographic information was collected in a pre-designed data sheet.
Standard procedures were followed for the collection, transport, processing, and culture of the urine samples. Samples once collected were sent to the laboratory immediately. From urine container, 0.01ml urine sample was inoculated immediately on a Cysteine Lactose Electrolyte Deficient (CLED) agar plate with the help of a calibrated double loop inoculator (Himedia, India). Plates were then incubated for 18-24 hours in an incubator at 37ºC. E. coli was identified by motility, sugar fermentation, methyl red, Voges Proskeuer, indole, citrate, urease, hydrogen sulfide formation, and oxidase tests [9]. E. coli isolates were tested for antibiotic susceptibility by Kirby Bauer disc diffusion method. Antibiotic discs used were gentamicin-10µg, amikacin-30µg, ampicillin-10µg, amoxicillin/clavulanic acid-20/10µg, ceftazidime-30µg, ceftriaxone-30µg, meropenem-10µg, piperacillin/tazobactam-100/10µg, nalidixic acid-30µg, norfloxacin-10µg, co-trimoxazole- 1.25/ 23.75µ, nitrofurantoin-300µg and fosfomycin-200 µg. The result was interpreted according to CLSI 2021 guidelines. For fosfomycin, the inhibition zone of >16mm, 13-15mm and <12mm was interpreted as sensitive, intermediate sensitive and resistant respectively according to CLSI 2021 guideline [10]. MDR was defined as resistance to a minimum one drug of three or more groups of antibiotics [11].
ESBL production in E. coli was detected by double disc synergy test (DDST) as described earlier [12]. Mueller Hinton agar was inoculated with standardized inoculums (corresponding to 0.5 McFarland tube) of test organism. An amoxicillin/clavulanic acid disc 20/10 μg was placed in the center of the plate and test discs of 3rd generation cephalosporins (ceftazidime- CAZ 30μg, ceftriaxone-CRO 30μg, cefotaxime-CTX 30μg) discs were placed at 20 mm distance (center to center) from the amoxicillin-clavulanic acid disc. The plate was incubated overnight at 35°C. Enhancement of the zone of inhibition of any one of the three drug discs toward amoxicillin-clavulanic acid suggested the presence of ESBLs. AmpC producers were detected by the cefoxitin-oxacillin disk diffusion test [13]. MBL detection was done by a combined disc test, in which imipenem and imipenem plus EDTA disc was used [14].
Results
During the study period, 88 E. coli were isolated. Out of them, 47 (53.40%) were from male patients and 41 (46.59%) were from female patients. Of the total samples, 30 (34.1%), 26 (29.55%) and 25 (28.41%) were from patients above 60, 18-40 and 41-60 years age group respectively. Out of 88 E. coli isolates, 58 (65.9%) were from in-patient cases (Table-1). Out of 58 urine samples from in-patient departments, only 4 were from intensive care unit (ICU).
Table-1: Distribution of gender, age and source of study cases (N=88)
Susceptibility of isolated E. coli to different antimicrobial agents is shown in Table-2. Highest resistance of E. coli was noted against nalidixic acid (93.8%) followed by ampicillin (81.82%), cephalosporins (77.27%) and norfloxacin (72.73%). Rate of resistance was low for nitrofurantoin (15.91%), piperacillin/tazobactam (17.05%), meropenem (18.18%), amoxycillin+ clavulanic acid (25%) and amikacin (23.86%). All the 88 (100%) isolated E. coli was sensitive to fosfomycin. Out of total E. coli, 78.4% was MDR strains.
Table-2: Susceptibility of isolated E. coli to fosfomycin and other antimicrobial agents (N=88)
Table-3 shows that out of 88 E. coli tested, 35 (39.8%) isolates were both AmpC and ESBLs producers, while 11 (12.5%) and 8 (9.1%) were only ESBL and AmpC producers respectively. MBL was positive in 15 (17%) E. coli isolates. All 69 ESBL, AmpC and MBL positive E. coli isolates were sensitive to fosfomycin.
Table-3: Distribution of ESBL, AmpC and MBL positive E. coli and their susceptibility to fosfomycin
Discussion
Urinary tract infection is a common problem in clinical practice. The study was conducted in a tertiary care setting in western Maharashtra, India to find out the prevalence of fosfomycin resistance among E. coli isolated from patients with UTI. UTI caused by a multi-drug resistant strain pose a serious challenge for the physician and also is a burden on the patient. Multidrug-resistant (MDR) isolates have emerged worldwide with the widespread use of cephalosporins and fluoroquinolones [15,16]. As a result, use of carbapenems has increased over the last 20 years, resulting into dramatic spread of carbapenem resistance [17,18]. Fosfomycin, discovered more than 40 years ago, is active against a wide range of organisms, including MDR Enterobacteriaceae [3,19,20].
Studies with fosfomycin are limited though, it is available for intravenous and oral use [19,21]. Recently, it has been shown to be non-inferior to piperacillin-tazobactam for the treatment of complicated urinary tract infections [22]. It is also been shown non-inferior to comparators for the treatment of bacteremic urinary tract infections due to MDR E coli [23].
In our study, a total of 88 isolates of E. coli were collected and analyzed. The majority of the urine samples were from male patients. Most isolates of E. coli were from UTI cases aged 60 and above and were from hospitalized patients (61%). About 78.4% of our E. coli isolates were MDR strains and positive for ESBL, AmpC or MBL. Niranjan et al found 38% of his E. coli isolates from UTIs were from in-patients and 76.5% of the isolates were MDR [24]. Hasan et al found 53% of E. coli from UTI cases as MDR strains [25]. Paul et al from Assam, India reported 26.2% ESBL and 12.6% carbapenemase producing E. coli from UTI cases [26]. All our E. coli isolates tested were sensitive to fosfomycin (100%). There was no difference in fosfomycin sensitivity between sensitive and MDR strains. Similar to our findings, Sabharwal et al in their study found 97% sensitivity to fosfomycin in E. coli isolated from UTI cases [27]. Our study has demonstrated that fosfomycin has excellent activity against MDR E. coli causing UTI in our area. Thus, the finding would help in formulating antibiotic treatment guideline for UTIs due to multi-resistant E. coli.
However, our study had some limitations. This study was conducted only for a short period of time with 88 E. coli isolates at a single center and minimum inhibitory concentration (MIC) of fosfomycin for those was not determined. Hence, multicenter studies with large sample size would provide a better perspective of the resistance pattern of uropathogenic E. coli to fosfomycin and other drugs in western part of Maharashtra.
Acknowledgement
The authors would also like to acknowledge the contributions of staff and laboratory personnel of the Department of Microbiology, Dr. D.Y. Patil Medical College, hospital and research center, Pimpri, Pune.
Conflict of interest: The authors have no conflict of interests to declare.
Fund: None
References
1. Bono MJ, Leslie SW, Reygaert WC. Urinary tract infection. [Updated 2022 Jun 15]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK470195/
2. Maraki S, Samonis G, Rafailid is PI, Vouloumanou EK, Mavromanolakis E, Falagas ME. Susceptibility of urinary tract bacteria to fosfomycin. Antimicrob Agents Chemother. 2009; 53(10): 4508-4510.
3. Dijkmans AC, Zacarías NVO, Burggraaf J, Mouton JW, Wilms EB, van Nieuwkoop C, et al. Fosfomycin: Pharmacological, Clinical and Future Perspectives. Antibiotics (Basel). 2017 Oct 31; 6(4): 24.
4. Sreenivasan S, Kali A, Pravin Charles MV, Kunigal S. Evaluation of in vitro susceptibility of fosfomycin among Enterobacteriaceae isolates from urine cultures: A study from Puducherry. J Lab physicians. 2019; 11(3): 249-252.
5. Ruiz Ramos J, Salavert Lletí M. Fosfomycin in infections caused by multidrug-resistant Gram-negative pathogens. Rev Esp QuiMioter. 2019; 32(Suppl 1): 45-54.
6. Brown ED, Vivas EI, Walsh CT, Kolter R. MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J Bacteriol. 1995; 177(14): 4194-7.
7. Reffert JL, Smith WJ. Fosfomycin for the treatment of resistant Gram-negative bacterial infections. Insights from the society of infectious diseases pharmacists. Pharmacotherapy. 2014; 34(8): 845-857.
8. Collee JG, Duguid JP Fraser AG, Marmion BP, Simmons A. Laboratory strategy in the diagnosis of infective syndromes. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie & McCartney Practical Medical Microbiology. 14th ed. India; Churchill Livingstone: 2011.
9. Winn, W., Allen, S., Janda, W., Koneman, E., Procop, G., Schreckenberger, P. and Woods, G. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 7th Edition, Lippincott Williams and Wilkins, New York. 2016; Page 225-255.
10. CLSI, Performance standards for antimicrobial susceptibility testing; 31st edition. CLSI Supplement M100. Clinical Laboratory Standard Institute 2021.
11. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18(3): 268-281.
12. Banerjee S, Sengupta M, Sarker TS. Fosfomycin susceptibility among multidrug-resistant, extended-spectrum beta-lactamase-producing, carbapenem-resistant uropathogens. Indian J Urol.2017; 33(2): 149-54.
13. Gopichand P, Agarwal G, Natarajan M, Mandal J, Deepanjali S, Parameswaran S, Dorairajan LN. In vitro effect of fosfomycin on multi-drug resistant gram-negative bacteria causing urinary tract infections. Infect Drug Resist. 2019 Jul 9; 12: 2005-2013.
14. Das NK, Grover N, Sriram R, Kumar M, Dudhat VL, Sarangan P. Prevalence of carbapenem resistance and comparison between different phenotypic methods for detection of metallo-beta-lactamases in Gram-negative non-fermentative bacteria in the acute wards of a tertiary care centre. Int J Curr Microbiol Appl Sci. 2016; 5(5): 109-119.
15. Rodríguez-Baño J, Pascual A. Clinical significance of extended-spectrum beta-lactamases. Expert Rev Anti Infect Ther. 2008; 6(5): 671-683.
16. Holland MS, Nobrega D, Peirano G, Naugler C, Church DL, Pitout JDD. Molecular epidemiology of Escherichia coli causing bloodstream infections in a centralized Canadian region: a population-based surveillance study. Clin Microbiol Infect. 2020; 26(11): 1554.e1-1554.e8.
17. Klein EY, Van Boeckel TP, Martinez EM, et al. Global increase and geographic convergence in antibiotic consumption between 2000 and 2015. Proc Natl Acad Sci U S A. 2018; 115(15): E3463-E3470.
18. Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: an evolving crisis of global dimensions. Clin Microbiol Rev. 2012; 25(4): 682-707.
19. Falagas ME, Vouloumanou EK, Samonis G, Vardakas KZ. Fosfomycin. Clin Microbiol Rev. 2016; 29(2): 321-347.
20. Falagas ME, Kastoris AC, Kapaskelis AM, Karageorgopoulos DE. Fosfomycin for the treatment of multidrug-resistant, including extended-spectrum beta-lactamase producing, Enterobacteriaceae infections: a systematic review. Lancet Infect Dis. 2010; 10(1): 43-50.
21. Grabein B, Graninger W, Rodríguez Baño J, Dinh A, Liesenfeld DB. Intravenous fosfomycin-back to the future: systematic review and meta-analysis of the clinical literature. Clin Microbiol Infect. 2017; 23(6): 363-372.
22. Kaye KS, Rice LB, Dane AL, et al. Fosfomycin for injection (ZTI-01) versus piperacillin-tazobactam for the treatment of complicated urinary tract infection including acute pyelonephritis: ZEUS, a phase 2/3 randomized trial. Clin Infect Dis. 2019; 69(12): 2045-2056.
23. Sojo-Dorado J, López-Hernández I , Rosso-Fernandez C, Morales IM, Palacios-Baena ZR, Hernández-Torres A, et al. Effectiveness of fosfomycin for multidrug-resistant urinary tract infections from E coli. JAMA Network Open. 2022; 5(1): e2137277.
24. Niranjan V, Malini A. Antimicrobial resistance pattern in Escherichia coli causing urinary tract infection among inpatients. Indian J Med Res. 2014; 139(6): 945-8.
25. Hasan AS, Nair D, Kaur J, Baweja G, Deb M, Aggarwal P. Resistance patterns of urinary isolates in a tertiary Indian hospital. J Ayub Med Coll Abbottabad. 2007; 19: 39–41.
26. Paul D, Anto N, Bhardwaj M, Prendiville A, Elangovan R, Bachmann TT, et al. Antimicrobial resistance in patients with suspected urinary tract infections in primary care in Assam, India. JAC-Antimicrobial Resistance. 2021; 3(4): dlab164.
27. Sabharwal ER, Sharma R. Fosfomycin: an alternative therapy for the treatment of UTI amidst escalating antimicrobial resistance. J Clin Diagn Res. 2015; 9(12): DC06-9.
Cite this article as:
Lohariwal Y, Das NK, Mirza S, Gandham N, Patil R, Mukhida, S, et al. Fosfomycin susceptibility among Escherichia coli causing urinary tract infection in a tertiary care centre in Western Maharashtra. IMC J Med Sci. 2023; 17(1): 008. DOI: https://doi.org/10.55010/imcjms.17.008
